Oxalic acid, a compound that is toxic to almost all organisms, is produced in large quantities by cellular metabolism. A number of pathological conditions can arise if oxalate accumulates in Man, including hyperoxaluria, the formation of calcium oxalate stones in the kidney (urolithiasis), renal failure, cardiomyopathy and cardiac conductance disorders. In addition, high levels of oxalate appear correlated with vulvodynia, a painful disease in women for which no treatment is currently available. Evidence has emerged to support the clinical application of oxalate-metabolizing enzymes in new, and intriguing, therapeutic strategies for lowering oxalate levels in biological fluids. The Yvrk gene found in Bacillus subtilis encodes oxalate decarboxylase (OxDC), an enzyme that converts oxalate to formate and CO2 in a Mn-dependent reaction for which the catalytic mechanism is not known. As part of our long-term aim to facilitate the use of OxDC in the treatment of oxalate-related illness, this project seeks to characterize Bacillus subtilis OxDC using the techniques of bioinorganic chemistry, molecular spectroscopy, enzyme kinetics and protein engineering. These studies are also likely to impact general understanding of radical mediated enzyme catalysis and to give new insights into (i) the role of protein environment in modulating metal reactivity and (ii) metalloenzyme evolution. Specific aims of this project are: 1) To investigate the catalytic mechanism of bacterial oxalate decarboxylase using steady-state kinetics, site-directed [unreadable] mutagenesis and isotope effects, 2) To determine the metal-dependence of OxDC, 3) To evaluate the effect of protein environment in controlling the chemical properties of the metal center(s) in OxDC, and 4) To identify steady-state radicals formed during steady-state turnover of oxalate decarboxylase. [unreadable] [unreadable] [unreadable]